Methods for nucleic acid manipulation

ABSTRACT

A method for replicating and amplifying a target nucleic acid sequence is described. A method of the invention involves the formation of a recombination intermediate without the prior denaturing of a nucleic acid duplex through the use of a recombination factor. The recombination intermediate is treated with a high fidelity polymerase to permit the replication and amplification of the target nucleic acid sequence. In preferred embodiments, the polymerase comprises a polymerase holoenzyme. In further preferred embodiments, the recombination factor is bacteriophage T4 UvsX protein or homologs from other species, and the polymerase holoenzyme comprises a polymerase enzyme, a clamp protein and a clamp loader protein, derived from viral, bacteriophage, prokaryotic, archaebacterial, or eukaryotic systems.

CROSS-REFERENCE TO RELATED APPLICATIONS

This Application claims priority of U.S. Provisional application Ser.No. 60/285,127 filed Apr. 20, 2001.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with United States Government support in theform of a grant from the National Institute of Health, Grant No.GM13306. The United States Government has certain rights in thisinvention.

REFERENCE TO A MICROFICHE APPENDIX

Not applicable

BACKGROUND OF THE INVENTION

The present invention is directed towards a process for theamplification of a target nucleic acid sequence contained in a largernucleic acid independent of using a thermocycle or a thermostablepolymerase. Unlike current technologies that employ a thermocycle andare therefore dependent upon a thermostable polymerase, the currentinvention allows for specific primer template association at a lowtemperature that will remain constant over the duration of the reaction.

A method for the site-specific amplification of a region of nucleic acidis described. Current amplification technology is based upon thePolymerase Chain Reaction (PCR). This PCR system can be thought of asinvolving three main components. First DNA oligonucleotidescomplementary to the flanking ends of the target sequence are required.These DNA oligonucleotides serve as primers for the initiation of DNAreplication by the second component of the system, a thermostable DNAdependent DNA polymerase, such as the Taq polymerase. The use of athermostable DNA polymerase is absolutely required in PCR so that thepolymerase activity can survive the third component, the thermal cycle.The thermal cycle uses high temperatures, usually 95 degrees Celsius, tomelt the target duplex DNA so that a subsequent annealing temperature,usually in the range of 50-60 degrees Celsius, permits the annealing ofthe primers to the appropriate locations flanking the target DNA.Following the annealing step, the thermal cycle incorporates apolymerization temperature, usually 72 degrees Celsius, which is theoptimal temperature of polymerization for the current thermostablepolymerases used in PCR.

The requirement of a thermal cycle to facilitate the annealing of theprimers flanking the target DNA to be amplified has several drawbacks.Primer annealing temperature is an important parameter in the success ofPCR amplification. The annealing temperature is characteristic for eacholigonucleotide: it is a function of the length and base composition ofthe primer as well as the ionic strength of the reaction buffer. Thetheoretical amplification value is never achieved in practice. Severalfactors prevent this from occurring, including: competition ofcomplementary daughter strands with primers for reannealing (i.e. twodaughter strands reannealing results in no amplification); loss ofenzyme activity due to thermal denaturation, especially in the latercycles; even without thermal denaturation, the amount of enzyme becomeslimiting due to molar target excess in later cycles (i.e. after 25-30cycles too many primers need extending); possible second site primerannealing and non-productive priming. Moreover, primers must avoidstretches of polybase sequences (e.g. poly dG) or repeating motifs—thesecan hybridize with inappropriate register on the template. Invertedrepeat sequences should also be avoided so as to prevent formation ofsecondary structure in the primer, which would prevent hybridization totemplate.

An additional drawback is the costly need for temperature baths, whichare required to shift their temperatures up and down rapidly, and in anautomated programmed manner. These are known as thermal cyclers or PCRmachines.

A further problem with PCR is the lack of fidelity of the variousPolymerases (Table 1) under different conditions. However, withincreasing number of cycles the greater the probability of generatingvarious artifacts (e.g. mispriming products). It is unusual to findprocedures that have more than 40 cycles. Errors made by DNA polymerasecan affect the extension. reaction of PCR during five distinct steps:(1) the binding of the correct dNTP by polymerase; (2) the rate ofphosphodiester bond formation; (3) the rate of pyrophosphate release;(4) the continuation of extension after misincorporation; and (5) theability of the enzyme to adjust to a misincorporated base by providing3′-to-5′ exonuclease ‘proofreading’ activity. Misincorporation rates fordifferent polymerases are described in terms of errors per nucleotidepolymerized, and the rate can be greatly affected by many parameters.Several studies have concluded that different thermostable DNApolymerases have error rates as high as 2.1×10⁻⁴ to 1.6×10⁻⁶ errors pernucleotide per extension (Table 2).

Another major drawback is that standard PCR protocols can amplify DNAsequences of 3000 base pairs (3 kb) or less. Efficient long PCR requiresthe use of two polymerases: a non-proofreading polymerase is the mainpolymerase in the reaction, and a proofreading polymerase (3′ to 5′ exo)is present at a lower concentration. Following the results of Cheng etal. the Tth enzyme (ABI/Perkin-Elmer) enzyme has been used as themain-component polymerase and Vent (New England Biolabs) as thefractional-component polymerase. Other combinations of proofreading andnon-proofreading polymerases are difficult to employ because differentactivities in specific buffer systems limits which combinations ofpolymerases can be used. Moreover, all of the problems associated withstandard PCR reactions become even more critical when attempting toamplify regions of DNA 3 kb or longer.

The current invention eliminates these problems with traditional PCR byeliminating the need for a thermal cycle and a thermostable polymerasein the amplification of a sequence of DNA embedded within a longertarget DNA. The current invention replaces the thermal cycle required toanneal the primers to the flanking ends of a target template byutilizing the enzymes active during homologous recombination, morespecifically during homologous pairing or D-loop formation.

In bacteriophage T4, DNA replication, as well as being initiated fromspecific origins of replication, is also very efficiently initiated fromrecombination intermediates. Therefore; the current invention isdirected at a system that primes DNA replication, in a specific manner,via recombination intermediates formed at opposite ends of a targetsequence embedded within a much larger sequence. This permits thereaction to be run at room temperature and therefore permits the use ofa non-thermal stable polymerase, The primary advantage of employing anon thermostable polymerase is that several polymerases have beencharacterized which have far superior fidelity. Moreover, thecharacterization of accessory factors, such as sliding clamp proteins,are known to increase the length of DNA which can be amplified to entiregenomes. In addition, the utilization of enzymes to deliver the primerseliminates all of the problems associated with annealing primers withinthe context of a thermal cycle mentioned above. Moreover, the homologouspairing reaction catalyzed by the bacteriophage T4 proteins is extremelyefficient and would eliminate the problem of mis-priming.

TABLE 1 Thermostable DNA polymerases and their sources DNA PolymeraseNatural or recombinant Source Taq Natural Thermus aquaticus Amplitaq ®Recombinant T. aquaticus Amplitaq (Stoffel Recombinant T. aquaticusfragment) ® Hot Tub ™ Natural Thermus flavis Pyrostate ™ Natural T.flavis Vent ™ Recombinant Thermococcus litoralis Deep Vent ™ RecombinantPyrococcus GB-D Tth Recombinant Thermus thermophilus Pfu NaturalPyrococcus furiosus ULTma ™ Recombinant Thermotoga maritima

TABLE 2 Properties of DNA polymerases used in PCR Taq/ Stoffel DeepAmplitaq ® fragment Vent ™ Vent ™ Pfu Tth ULTma ™ 95° C. half- 40 min 80min 400 min 1380 min >120 min 20 min >50 min life 5′3′ exo + − − − − + −3′5′ exo − − + + + − + Extension 75 >50 >80 ? 60 >33 ? rate (nt/sec) RTactivity Weak Weak ? ? ? Yes ? Resulting 3′ A 3′ A >95% >95% blunt 3′ Ablunt ends blunt blunt Strand − − + + − − − displacement M.W. (kDa) 94  61 ? ? 92   94 70

Error Rates

-   1. Taq (Thermus aquaticus)

1.1×10⁻⁴ base substitutions/bp [Tindall, K. R., and Kunkel, T. A. (1988)Biochemistry 27, p 6008-6013, “Fidelity of DNA synthesis by the Thermusaquaticus DNA polymerase.”]

2.4×10⁻⁵ frameshift mutations/bp [Tindall and Kunkel, Id.]

2.1×10⁻⁴ errors/bp [Keohavong, P., and Thilly, W. G. (1989) Proc NatlAcad Sci USA 86(23), p 9253-9257, “Fidelity of DNA polymerases in DNAamplification.”]

7.2×10−5 errors/bp [Ling, L. L., Keohavong, P., Dias, C., and Thilly, W.G. (1991) PCR Methods Appl 1(1) p 63-69, “Optimization of the polymerasechain reaction with regard to fidelity: modified T7, Taq, and Vent DNApolymerases.”]

8.9×10⁻⁵ errors/bp [Cariello, N. F., Swenberg, J. A., and Skopek, T. R.(1991) Nucleic Acids Res 19(15), p 4193-4198, “Fidelity of ThermococcusLitoralis DNA Polymerase (Vent) in PCR determined by denaturing gradientgel electrophoresis.”]

2.0×10⁻⁵ errors/bp [Lundberg, K. S., Shoemaker, D. D., Adams, M. W.,Short, J. M., Sorge, J. A., and Mathur, E. J. (1991) Gene 108(1), p 1-6,“High-fidelity amplification using a thermostable DNA polymeraseisolated from Pyrococcus furiosus.”]

1.1×10⁻⁴ errors/bp [Barnes, W. M. (1992) Gene 112(1), p 29-35, “TheFidelity of Taq polymerase catalyzing PCR is improved by an N-terminaldeletion.”]

-   2. KlenTaq (Thermus aquaticus, N-terminal deletion mutant)

5.1×10⁻⁵ errors/bp [Barnes, W. M. (1992) Gene 112(1), p 29-35, “TheFidelity of Taq polymerase catalyzing PCR is improved by an N-terminaldeletion.”]

-   3. Vent (Thermococcus litoralis)

2.4×10⁻⁵ errors/bp [Cariello, N. F., Swenberg, J. A., and Skopek, T. R.(1991) Nucleic Acids Res 19(15), p 4193-4198, “Fidelity of ThermococcusLitoralis DNA Polymerase (Vent) in PCR determined by denaturing gradientgel electrophoresis.”]

4.5×10⁻⁵ errors/bp [Ling, L. L., Keohavong, P., Dias, C., and Thilly, W.G. (1991) PCR Methods Appl 1(1) p 63-69, “Optimization of the polymerasechain reaction with regard to fidelity: modified T7, Taq, and Vent DNApolymerases.”]

5.7×10-5 errors/bp [Matilla, P., Korpela, J., Tenkanen, T., andPitkanen, K. (1991) Nucleic Acids Res 19(18), p 4967-4973, “Fidelity ofDNA synthesis by the Thermococcus litoralis DNA polymerase—an extremelyheat stable enzyme with proofreading activity.”]

-   4. Vent(exo-) (Thermococcus litoralis)

1.9×10⁻⁴ errors/bp [Matilla et al., Id.]

-   5. Deep Vent (Pyrococcus species GB-D)

No published literature. New England Biolabs claims fidelity is equal toor greater than that of Vent.

-   6. Deep Vent(exo-)

No published literature.

-   7. Pfu (Pyrococcus furiosus)

1.6×10⁻⁶ errors/base [Lundberg, K. S., Shoemaker, D. D., Adams, M. W.,Short, J. M., Sorge, J. A., and Mathur, E. J. (1991) Gene 108(1), p 1-6,“High-fidelity amplification using a thermostable DNA polymeraseisolated from Pyrococcus furiosus.”]

-   8. Replinase (Thermus flavis)

1.03×10⁻⁴ errors/base [Matilla, P., Korpela, J., Tenkanen, T., andPitkanen, K. (1991) Nucleic Acids Res 19(18), p 4967-4973, “Fidelity ofDNA synthesis by the Thermococcus litoralis DNA polymerase—an extremelyheat stable enzyme with proofreading activity.”]

BRIEF SUMMARY OF THE INVENTION

In one aspect, the present invention contemplates a method forreplicating and amplifying a target nucleic acid sequence comprisingreacting a primer that is complementary to a target sequence within anucleic acid duplex with the nucleic acid duplex in the presence of arecombination factor to form a recombination intermediate, withoutpreviously denaturing the nucleic acid duplex. The recombinationintermediate so formed is then admixed with a polymerase to form apolymerase complex, whereby the polymerase replicates the targetsequence. Preferably, the polymerase is a polymerase holoenzyme. Morepreferably, the polymerase holoenzyme comprises a polymerase enzyme, aclamp protein, and a clamp loader protein. Although other sources andmaterials can be used, it is preferred that the recombination factor,the polymerase, the clamp protein and clamp loader be obtained frombacteriophage T4. Thus, the recombination factor is preferablybacteriophage T4 UvsX protein, the polymerase is preferablybacteriophage T4 gene product (gp) 43 polymerase, the clamp protein ispreferably bacteriophage T4 gp 45 clamp protein and the clamp loader ispreferably bacteriophage T4 gp44/gp 62 clamp loader complex.

In one preferred embodiment, the primer is designed to anneal atcomplimentary sites flanking the target nucleic acid sequence. In afurther preferred embodiment, the polymerase holoenzyme complexcomprises a viral, bacteriophage, eukaryotic, archaebaoterial, orprokaryotic polymerase holoenzyme complex. Preferably, the bacteriophageis T4, and the holoenzyme complex includes the gene product 43polymerase. Preferably, the bacteriophage is T4, and the holoenzymecomplex includes the gene product 45 clamp protein.

In a further aspect, a contemplated method uses a single strandedbinding protein to facilitate downstream strand displacement synthesisby the polymerase holoenzyme complex. The single stranded protein ispreferably gene product 32 from the T4 bacteriophage system.

In yet another aspect, a contemplated method uses a single strandedbinding protein to destabilize the helix at or near the point of theprimer template junction.

In a still further aspect, the present invention contemplates a methodfor reproducing and amplifying a target nucleic acid sequence at atemperature below about 45° Celsius and comprises catalyticallyinserting a primer into the target nucleic acid sequence withoutpreviously denaturing the duplex in whole or in part to form arecombination intermediate. The recombination intermediate so formed isthen admixed with a polymerase to form a polymerase complex, whereby thepolymerase replicates the target nucleic acid sequence. The primer ispreferably pretreated with single stranded nucleic acid binding protein.It is also preferred that the primer be pretreated with a recombinationfactor. A preferred recombination factor is bacteriophage T4 UvsX. Apreferred polymerase is gene product 43 DNA polymerase from thebacteriophage T4.

In a further aspect, an above-contemplated method uses a helicase tofacilitate replication by the polymerase. A preferred helicase isbacteriophage T4 gene product 41 DNA helicase. A further preferredhelicase is bacteriophage T4 replicative helicase complex, comprisingbacteriophage T4 gp 41 and gp 59.

In another aspect, a contemplated method uses an accessory factor tostabilize the recombination factor. A preferred accessory factor isbacteriophage T4 UvsY. In a still further aspect, a contemplated methoduses a combination of a helicase and an accessory factor. A preferredhelicase is bacteriophage T4 gp41. A preferred accessory factor isbacteriophage T4 UvsY.

In yet another aspect, the present invention contemplates a method ofcreating a library of nucleic acid sequences. This method comprisesincubating a first double-stranded nucleic acid with an enzyme withexonuclease activity to form a plurality of single stranded DNA regionshaving random sizes. This plurality of single stranded DNA regions istreated with a recombination factor to form a plurality of pretreatedsingle stranded DNA regions. A second double-stranded nucleic acid isthen added to the plurality of pretreated single stranded DNA regions toform a plurality of three stranded crossover junctions. The plurality ofthree stranded crossover junctions is incubated with a helicase to forma plurality of Holliday junctions. The phirality of Holliday junctionsso formed is resolved by incubation with an endonuclease. Preferably,the recombination factor is bacteriophage T4 UvsX. Preferably, thehelicase is bacteriophage T4 gene products 41 and 59, A furtherpreferred helicase is bacteriophage T4 UvsW. A preferred endonuclease isbacteriophage T4 gp 49.

BRIEF DESCRIPTION OF THE DRAWINGS

In the drawings forming a portion of this disclosure:

FIG. 1 depicts polymerase holoenzyme complex formation at arecombination D-loop.

FIG. 2 depicts a gene amplification system using a method of theinvention.

FIG. 3 depicts creation of a library of novel recombinant nucleic acidsequences using a method of the invention.

DETAILED DESCRIPTION OF THE INVENTION A. Basic Replication andAmplification Process

In one aspect, a process of the invention comprises treating a nucleicacid, such as RNA or DNA, with an oligonucleotide primer, which primeris complementary to a predetermined target sequence within that nucleicacid. Preferably, the nucleic acid is double stranded, such as in theform of a DNA heteroduplex. A process of the invention contemplatesreacting separate complementary strands of a nucleic acid heteroduplexwith a molar excess of two oligonucleotide primers. Significantly, thistreatment does not require the prior denaturation of the complementarystrands of the nucleic acid heteroduplex. Rather, the hybridization ofthe primer with its target sequence is mediated by a recombinationfactor. The recombination factor functions to form a recombinationintermediate. An exemplary recombination intermediate is a D-loopstructure between the primer and the complementary strands of thenucleic acid heteroduplex. The recombination factor can be used topre-treat the primer at temperatures below 90° C., more preferably below45° C., and most preferably at 37° C.

A preferred recombination factor is the bacteriophage T4 UvsX geneproduct. A recombination factors, such as UvsX, can require additionalcomponents, such as ATP, in order to optimally function. Thebacteriophage T4 UvsX protein functions to facilitate formation of apresynaptic filament capable of undergoing homologous pairing (See FIG.1). When mixed with the target nucleic acid, the resultant recombinationintermediates, positioned at opposite ends of the target nucleic acid,can serve as sites for attachment of a polymerase, A preferredpolymerase is a polymerase holoenzyme. Preferably, the polymeraseholoenzyme is the polymerase holoenzyme of bacteriophage T4.

The formation of the bacteriophage T4 polymerase holoenzyme complex isshown in FIG. 2, This polymerase holoenzyme complex includes the geneproduct (gp) 43 DNA polymerase, the gene product 45 clamp protein, andthe gene products 44 and 62, which together facilitate association ofthe gene 45 clamp with the gene product 43 polymerase explicitly at therecombination intermediate primer/template junction (See FIG. 2).

The gene product 32 single-stranded binding protein is added tofacilitate strand displacement synthesis by the polymerase holoenzymecomplex (See FIG. 2). The addition of accessory factors to stabilize therecombination factor is also contemplated. For example, thebacteriophage UvsY protein, a UvsX protein accessory factor, serves tostabilize the initial presynaptic filament permitting the introductionof the bacteriophage T4 replicative helicase complex, the products ofthe genes 41 and 59. This will constitute the assembly of an intactreplication fork which can extend the range of site specific nucleicacid amplification beyond what can be expected using availablethermostable polymerases alone during a thermocycle.

In some embodiments of a. method of the invention, a polymerase withhigh fidelity and high processivity is used, namely bacteriophage T4 gp43 DNA polymerase. This polymerase has been shown to have an accuracy inreplication that is orders of magnitude greater than those polymerasescommonly associated with PCR, and with the PCR technique. Thispolymerase has a built in proofreading/editing function which, when usedin connection with a self contained DNA duplication process,significantly increases the accuracy of that duplication process.

Not only does this increase in processivity produce a more accurateduplicate, it enhances the ability of the technique to accuratelyreplicate target nucleic acids with many thousands of base pairs.Homologous polymerase holoenzymes are found in other species, such asthe DNA polymerase III found in prokaryotic systems, and DNA polymerasedelta and epsilon holoenzymes in eukaryotic systems.

In other embodiments of a method of the invention, use of a holoenzymecomplex, a gene clamp, and a helicase complex, enables polymerase toefficiently operate along much longer nucleic acid sequences than canpredictably be duplicated with existing PCR technologies.

In a further aspect, the present invention contemplates a method forreproducing and amplifying a target nucleic acid sequence at atemperature below about 45° Celsius and comprises catalyticallyinserting a primer into the target nucleic acid sequence withoutpreviously denaturing the duplex in whole or in part to form arecombination intermediate. The entire duplication and amplificationprocess can occur at temperatures below about 90° C., more preferablybelow about 45° C., and most preferably at about 37° C., due to thecatalytic nature of the involved processes. Because no thermostableenzymes are required, the problems associated with magnesium chloridesolutions and their concentrations are avoided. Similarly, the use ofmineral oil is eliminated.

Because the present invention is based upon the creation of arecombination intermediate, such as a D-loop, without previouslydenaturing the duplex, and can be conducted at temperatures of about 37°C., it eliminates the extraneous and undesirable/nonspecific annealingthat occurs along the length of the denatured duplex, and eliminatesissues of having the wrong primer concentrations, programmingdifficulties with PCR machines, and having excessive or insufficienttemplates.

Avoiding these problems commonly associated with PCR further augmentsthe capability of a process of the invention to replicate and amplifywith greater fidelity and processivity.

The recombination intermediate so formed is then admixed with apolymerase to form a polymerase complex, whereby the polymerasereplicates the target nucleic acid sequence. The primer is preferablypretreated with single stranded nucleic acid binding protein. It is alsopreferred that the primer be pretreated with a recombination factor. Apreferred recombination factor is bacteriophage T4 UvsX, A preferredpolymerase is gene product 43 DNA polymerase from the bacteriophage T4.

As is understood by one of ordinary skill in the art, the replicationand amplification of a target nucleic acid sequence by a polymeraserequires the presence of nucleotide triphosphates in concentrationssufficient to permit elongation of the nascent copies. In addition, theconcentrations of the other components of a method of the invention canreadily be determined by one of ordinary skill in the art, based uponempirical determinations as well as the examples that follow.

Moreover, as is well understood by one of ordinary skill in the art, amethod of the present invention permits additional rounds, or cycles, ofreplication of a target nucleic acid sequence, by virtue of there-initiation of a method of the invention. As such, not only is atarget nucleic acid sequence copied or replicated, it is amplified as aresult of the repetition of a method of the invention. While not wishingto be bound by theory, it is believed that the presence of a molarexcess of a primer in embodiments of the invention permits the repeatedformation of a recombination intermediate and subsequent replication ofa nucleic acid target. In this sense, the present invention provides analternative to the target amplification of PCR.

B. Use of Process to Create Libraries

In addition to specific nucleic acid amplification, a process of theinvention contemplates the use of the bacteriophage T4 presynapticfilament (gene products of the UvsX, UvsY, and gp32 genes) to promotethe recombination of different nucleic acid sequences to produce aprotein with desired novel functional characteristics. This methodcomprises incubating a first double-stranded nucleic acid with an enzymehaving exonuclease activity to form a plurality of single stranded DNAregions having random sizes. The exonuclease treatment is performedunder conditions that would randomize the size and distribution of theresultant single stranded DNA region.

This plurality of single stranded DNA regions is treated with arecombination factor to form a plurality of pretreated single strandedDNA regions. For example, in a preferred embodiment, in the presence ofthe bacteriophage T4 UvsX and UvsY gene products and a second undigestedtarget nucleic acid sequence, an initial three stranded crossoverreaction occurs in a random manner as dictated by the distribution ofthe exonuclease digestion of the first nucleic acid upon which thepresynaptic filament is formed.

A second double-stranded nucleic acid is then added to the plurality ofpretreated single stranded DNA regions to form a plurality of threestranded crossover junctions. The plurality of three stranded crossoverjunctions is incubated with a helicase to form a plurality of Hollidayjunctions. The plurality of Holliday junctions so formed is resolved byincubation with an endonuclease. Preferably, the recombination factor isbacteriophage T4 UvsX. Preferably, the helicase is bacteriophage T4 geneproducts 41 and 59. A further preferred helicase is bacteriophage T4UvsW. A preferred endonuclease is bacteriophage T4 gp 49.

Upon addition of helicase activity derived from the products of genes 41and 59, branch migration extends regions of heteroduplex DNA beyondregions of non- or partial homology. The final products of the reactioncan be resolved with the bacteriophage gene product 49 protein, anendonuclease that will specifically recognize and cleave recombinationcrossover junctions (Holliday junctions) (See FIG. 3).

Enzymes from other species can be used in a contemplated method of theinvention, in addition to enzymes from the bacteriophage T4 system. Forexample, enzymes from E. coli, including RecA, RecF, RecO, RecR, RuvA,RuvB, RecG, and RuvC, can be used, A recombination factor useful in someembodiments of a method of the invention includes the UvsX protein frombacteriophage T4, the RecA protein from E. coli, and the Rad51 proteinfrom yeast, as well as Rad51 homologs from other eukaryotic species. Anaccessory factor useful in some embodiments of a method of the inventionincludes the UvsY protein from bacteriophage T4, the Rec F, O and Rproteins from E. coli, and the Rad52 protein from yeast, as well asRad52 homologs from other eukaryotic species.

In addition to the bacteriophage T4 polymerase, clamp and clamp loadingcomplex, the DNA polymerase In holoenzyme, the beta-clamp clamp and thegamma-complex clamp loading complex from prokaryotic species can be usedin a method of the invention. Still further, the DNA polymerase deltaand epsilon holoenzymes, the PCNA clamp, and the replication factor Ccomplex clamp loading complex from eukaryotic species can be used in amethod of the invention.

EXAMPLES Example 1

Homologous recombination directed nucleic acid amplification of closedcircular plasmid DNA using the T4 holoenzyme complex and the T4homologous recombination proteins UvsX and gene product 32 wereperformed as follows. Oligonucleotide primers were designed to amplify a3220 base pair fragment from M13mp18 plasmid DNA as follows:

1940- (SEQ ID. NO. 1) 5′TGATACACCTATTCCGGGCTATACTTATAT-3′ and 5160(SEQ ID. NO. 2) 5′-CGCTCAATCGTCTGAAATGGATTATTTACATTGGCAGATT-3′.

These primers were used for the amplification of a 3220 base pairfragment from closed circular M13mp18 plasmid DNA. Reaction conditionswere set up to facilitate the assembly of the polymerase holoenzyme,including bacteriophage T4 gene products 43, 45, and 44/62, onrecombination intermediates formed by the action of bacteriophage T4UvsX protein. The concentration of double stranded closed circularM13mp18 plasmid DNA was set at 10 micrograms per milliliter (2.1nanomolar as nucleotides). Both oligonucleotides, 1940 (SEQ ID NO. 1)and 5160 (SEQ ID NO. 2), were used at a concentration of 210 nanomolar(as molecules). The concentration of UvsX was present to ensure about50% coverage of the 40mer oligonucleotide primers (UvsX monomer/4 basesite size), The concentration of ATP was set at 2 millimolar during theD-loop reaction, and an ATP regeneration system was employed consistingof phosphocreatine kinase and phosphoenol pyruvate. The gene product(gp) 32 single stranded protein was present at 25 micromolar. Theholoenzyme was constructed using 1 micromolar gp43 (polymerase), 1micromolar gp45 (as trimers, sliding clamp) and 500 nanomolar gp44/62complex (ATP dependent clamp loader). The deoxyribonucleotides werepresent at 3 millimolar.

The reaction order was designed to allow for the initial formation of arecombination intermediate, or D-loop, followed by the formation of apolymerase holoenzyme complex. The M13mp18 closed circular doublestranded DNA template was first incubated in holoenzyme complexformation buffer (20 millimolar Tris (pH7.5), 150 millimolar KOAc, 10millimolar Mg(OAc)₂) in the presence of the primers, 2 millimolar ATP,phosphoenol pyruvate and pyruvate kinase.

Homologous pairing, or D-loop-formation, was then initiated by theaddition of the T4 UvsX strand tranferase protein. After 2 minutes at37° C., an additional 1 millimolar ATP, 3 millimolar deoxyribonucleotidemix, gp 32 single stranded binding protein and the gp43 polymerase andthe gp45 and gp44/62 accessory factors were added to initiate polymeraseholoenzyme formation at recombination intermediates and initiate stranddisplacement DNA synthesis. At 10, 20 and 30 minutes aliquots of thereaction mix were removed and quenched with SDS and EDTA followed byheating to 60° C. for 10 minutes, The reactions were then loaded onto a1.0% TBE agarose gel and visualized using ethidium bromide.

Example 2

Homologous recombination directed nucleic acid amplification of linearplasmid DNA using the T4 holoenzyme complex and the T4 homologousrecombination proteins UvsX and gene product 32. M13mp18 double strandedclosed circular plasmid DNA was made linear by digestion with the BamH1restriction endonuolease. 10 micrograms M13mp18 and the BamH1restriction endonuclease were incubated at 37° C. using standard bufferconditions for 2 hours followed by phenol/chloroform extraction andpassage through two G-25 spin columns. Reaction conditions were asdescribed for Example 1.

Each of the patents and articles cited herein is incorporated byreference. The use of the article “a” or “an” is intended to include oneor more.

The foregoing description and the examples are intended as illustrativeand are not to be taken as limiting. Still other variations within thespirit and scope of this invention are possible and will readily presentthemselves to those skilled in the art.

1.-22. (canceled)
 23. A method for amplifying a target nucleic acidsequence within a nucleic acid molecule, comprising: providing a targetnucleic acid molecule containing a target nucleic acid sequence;forming, within a reaction vessel, a reaction mixture containing thetarget nucleic acid molecule, a UvsX recombination factor, two primersthat bind to the target nucleic acid sequence, a polymerase, andnucleotides in an amount sufficient to support amplification of thetarget nucleic acid sequence; and forming an amplification product byamplifying the target nucleic acid sequence within the reaction vessel.24. The method according to claim 1, further including contacting theamplification product with a fluorescent molecule.
 25. The methodaccording to claim 1, wherein the method further includes detecting theamplification product.
 26. The method according to claim 3, furtherincluding contacting the amplification product with a fluorescentmolecule.
 27. The method according to claim 4, wherein the detectingstep includes detecting fluorescence.
 28. The method according to claim1 wherein said polymerase is a holoenzyme complex.
 29. The methodaccording to claim 5, wherein said holoenzyme complex is a gene productof a viral, bacteriophage, prokaryotic, or eukaryotic system.
 30. Themethod according to claim 1 wherein the reaction vessel also includes asingle stranded nucleic acid binding protein.
 31. The method accordingto claim 1 wherein the reaction vessel also includes a helicase.
 32. Themethod according to claim 1 wherein the reaction vessel also includes anATP regeneration system.
 33. The method according to claim 1, furthercomprising forming a recombination intermediate.
 34. The methodaccording to claim 12, wherein forming the recombination intermediateincludes contacting the target nucleic acid sequence with the UvsXrecombination factor.
 35. The method according to claim 1, furtherincluding separating the amplification product from the reactionmixture.
 36. The method according to claim 1, further includinganalyzing the amplification product to confirm the identity of thetarget nucleic acid sequence.
 37. The method according to claim 1wherein the target nucleic acid molecule is double stranded.
 38. Themethod according to claim 1, wherein the target nucleic acid sequence isembedded within the longer target nucleic acid molecule.